Ceramide constitutes a family of closely related molecules that function as bioeffector lipids with roles in the regulation of stress responses and growth/death of various human cancer cells. Our laboratory has focused on defining specific pathways of ceramide metabolism and hence function. Studies from this project have defined a novel pathway for the generation of ceramide involving glucosylcerebrosidase (GBA1), a gene responsible for Gaucher's disease. Our preliminary data suggest that this specific pathway regulates the production of the pro-inflammatory IL-6 in breast cancer cells in response to cytokines through activation of the ceramide activated protein phosphatase (CAPP). These results have led us to the hypothesis that the novel salvage pathway involving GBA1/ceramide/CAPP functions to regulate the activity of p38 MAPK, which in turn couples the GBA/ceramide pathway to the regulation of cytokine-induced inflammatory responses, e.g. induction of IL- 6. Therefore, 3 specific aims are proposed: 1) To determine the role and regulation of the novel GBA1/salvage pathway in ceramide formation by a) establishing activation of the salvage pathway and the role of GBA1;b) determining the subcellular localization of GBA1;c) determining the roles of subsequent enzymes of sphingolipid metabolism in the salvage pathway, and d) defining upstream mechanisms of regulation of this pathway. 2) To determine the role of the GBA1/ceramide pathway in regulation of CAPP/p38 delta MAPK by a) establishing the regulation of p38 by GBA1;b) determining the specific lipid involved;c) determining the specific isoform of p38 involved in the process;d) determining the role of CAPP in mediating the effects of ceramide on p38 phosphorylation, the specific catalytic subunit, and the specific regulatory subunits;and e) determining the mechanisms of regulation of CAPP/p38 in vitro and in cells 3) To determine the role of the GBA1/ceramide/CAPP/p38 delta pathway in regulation of inflammatory responses, by a) determining the role and selectivity of this pathway in regulating IL-6 and other inflammatory mediators;and b) determining the role of this pathway in breast cancer progression/metastasis. These studies are beginning to define a novel pathway in the regulation of ceramide metabolism, mechanisms of action of ceramide, and an emerging role for ceramide in regulation of inflammatory responses. In addition, the studies are beginning to point to a heretofore unappreciated role for both GBA1 and p38 delta MAPK as key regulators of inflammatory responses. These results should be of great significance to cancer biology since ceramide may function as a tumor suppressor lipid, GBA1-defcient patients have increased risk of multiple myeloma and other cancers, including breast, IL-6 is serves as an etiologic factor in multiple myeloma and as an established poor prognostic marker for breast cancer and potential regulator of tumor metastasis, and p38 as a potential novel therapeutic target. PUBLIC HEALTH RELEVANCE: Bioactive sphingolipids are a class of lipid (fatty) molecules which are emerging as important regulators of cell functions, including effects on cell growth, cell death, and cell migration. The bioactive sphingolipid ceramide is a key molecule in sphingolipid metabolism and function. Our studies are beginning to implicate this lipid in regulating cell growth and death as well as inflammatory responses. In this proposal, we focus on a novel pathway by which this lipid is regulated. We also focus on the mechanisms by which this pathway regulates cancer cell responses to inflammatory agents. By dissecting out the molecular mechanisms of this pathway, we stand to not only advance our basic science understanding of how these molecules operate, but we anticipate to advance novel targets for cancer therapy as well as for the treatment of Gaucher disease.